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1.
Genome Biol ; 23(1): 189, 2022 09 07.
Article in English | MEDLINE | ID: mdl-36071529

ABSTRACT

BACKGROUND: A major challenge in neurodegenerative diseases concerns identifying biological disease signatures that track with disease progression or respond to an intervention. Several clinical trials in Huntington disease (HD), an inherited, progressive neurodegenerative disease, are currently ongoing. Therefore, we examine whether peripheral tissues can serve as a source of readily accessible biological signatures at the RNA and protein level in HD patients. RESULTS: We generate large, high-quality human datasets from skeletal muscle, skin and adipose tissue to probe molecular changes in human premanifest and early manifest HD patients-those most likely involved in clinical trials. The analysis of the transcriptomics and proteomics data shows robust, stage-dependent dysregulation. Gene ontology analysis confirms the involvement of inflammation and energy metabolism in peripheral HD pathogenesis. Furthermore, we observe changes in the homeostasis of extracellular vesicles, where we find consistent changes of genes and proteins involved in this process. In-depth single nucleotide polymorphism data across the HTT gene are derived from the generated primary cell lines. CONCLUSIONS: Our 'omics data document the involvement of inflammation, energy metabolism, and extracellular vesicle homeostasis. This demonstrates the potential to identify biological signatures from peripheral tissues in HD suitable as biomarkers in clinical trials. The generated data, complemented by the primary cell lines established from peripheral tissues, and a large panel of iPSC lines that can serve as human models of HD are a valuable and unique resource to advance the current understanding of molecular mechanisms driving HD pathogenesis.


Subject(s)
Huntington Disease , Neurodegenerative Diseases , Energy Metabolism , Humans , Huntington Disease/genetics , Inflammation/complications , Proteomics
2.
Stem Cell Res ; 53: 102276, 2021 05.
Article in English | MEDLINE | ID: mdl-33714067

ABSTRACT

CDKL5 Deficiency Disorder (CDD) is a rare X-linked monogenic developmental encephalopathy that is estimated to affect 1:42,000 live births. CDD is caused by pathogenic variants in the CDKL5 gene and is observed in both male and female patients. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts of six unrelated CDD patients-three males and three females. These patients are clinically diagnosed to present with classic CDD phenotypes, including refractory epilepsy and global developmental delay, and are being followed in a longitudinal clinical study.


Subject(s)
Epileptic Syndromes , Induced Pluripotent Stem Cells , Spasms, Infantile , Female , Humans , Male , Protein Serine-Threonine Kinases/genetics , Spasms, Infantile/genetics
3.
J Huntingtons Dis ; 9(1): 13-31, 2020.
Article in English | MEDLINE | ID: mdl-32007959

ABSTRACT

BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disorder that prominently affects the basal ganglia, leading to affective, cognitive, behavioral, and motor decline. The primary site of neuron loss in HD is the striatal part of the basal ganglia, with GABAergic medium size spiny neurons (MSNs) being nearly completely lost in advanced HD. OBJECTIVE: Based on the hypothesis that mutant huntingtin (mHTT) protein injures neurons via transcriptional dysregulation, we set out to establish a transcriptional profile of HD disease progression in the well characterized transgenic mouse model, R6/2, and two Knock-in models (KI); zQ175KI (expressing mutant mouse/human chimeric Htt protein) and HdhQ200 HET KI (carrying one allele of expanded mouse CAG repeats). METHODS: In this study, we used quantitative PCR (qPCR) to evaluate striatal mRNA levels of markers of neurotransmission, neuroinflammation, and energy metabolism. RESULTS: After analyzing and comparing transcripts from pre-symptomatic and symptomatic stages, markers expressed in the basal ganglia MSNs, which are typically involved in maintaining normal neurotransmission, showed a genotype-specific decrease in mRNA expression in a pattern consistent with human studies. In contrast, transcripts associated with neuroinflammation and energy metabolism were mostly unaffected in these animal models of HD. CONCLUSION: Our results show that transcripts linked to neurotransmission are significantly reduced and are consistent with disease progression in both zQ175KI and R6/2 transgenic mouse models.


Subject(s)
Corpus Striatum/metabolism , Disease Progression , GABAergic Neurons/pathology , Huntingtin Protein/metabolism , Huntington Disease/metabolism , Inflammation/metabolism , Mutant Proteins/metabolism , RNA, Messenger/metabolism , Transcription, Genetic/physiology , Animals , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Mutant Chimeric Proteins , Real-Time Polymerase Chain Reaction
4.
Epilepsia ; 61(2): e7-e12, 2020 02.
Article in English | MEDLINE | ID: mdl-31883345

ABSTRACT

Seizure prediction is feasible, but greater accuracy is needed to make seizure prediction clinically viable across a large group of patients. Recent work crowdsourced state-of-the-art prediction algorithms in a worldwide competition, yielding improvements in seizure prediction performance for patients whose seizures were previously found hard to anticipate. The aim of the current analysis was to explore potential performance improvements using an ensemble of the top competition algorithms. The results suggest that minor increments in performance may be possible; however, the outcomes of statistical testing limit the confidence in these increments. Our results suggest that for the specific algorithms, evaluation framework, and data considered here, incremental improvements are achievable but there may be upper bounds on machine learning-based seizure prediction performance for some patients whose seizures are challenging to predict. Other more tailored approaches that, for example, take into account a deeper understanding of preictal mechanisms, patient-specific sleep-wake rhythms, or novel measurement approaches, may still offer further gains for these types of patients.


Subject(s)
Algorithms , Electrocorticography/methods , Seizures/diagnosis , Crowdsourcing , Drug Resistant Epilepsy/diagnosis , Electroencephalography , Epilepsies, Partial/diagnosis , Feasibility Studies , Female , Humans , Machine Learning , Male , Middle Aged , Predictive Value of Tests , Reproducibility of Results , Sensitivity and Specificity , Sleep , Young Adult
5.
Biochem Biophys Res Commun ; 521(3): 549-554, 2020 01 15.
Article in English | MEDLINE | ID: mdl-31677786

ABSTRACT

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD.


Subject(s)
Huntingtin Protein/analysis , Animals , Cells, Cultured , Gene Knock-In Techniques , HEK293 Cells , Humans , Huntingtin Protein/genetics , Huntington Disease/genetics , Mice , Mutation , Neurons/chemistry , Neurons/metabolism , Phosphorylation , Protein Aggregates , Protein Processing, Post-Translational
6.
Brain ; 141(9): 2619-2630, 2018 09 01.
Article in English | MEDLINE | ID: mdl-30101347

ABSTRACT

Accurate seizure prediction will transform epilepsy management by offering warnings to patients or triggering interventions. However, state-of-the-art algorithm design relies on accessing adequate long-term data. Crowd-sourcing ecosystems leverage quality data to enable cost-effective, rapid development of predictive algorithms. A crowd-sourcing ecosystem for seizure prediction is presented involving an international competition, a follow-up held-out data evaluation, and an online platform, Epilepsyecosystem.org, for yielding further improvements in prediction performance. Crowd-sourced algorithms were obtained via the 'Melbourne-University AES-MathWorks-NIH Seizure Prediction Challenge' conducted at kaggle.com. Long-term continuous intracranial electroencephalography (iEEG) data (442 days of recordings and 211 lead seizures per patient) from prediction-resistant patients who had the lowest seizure prediction performances from the NeuroVista Seizure Advisory System clinical trial were analysed. Contestants (646 individuals in 478 teams) from around the world developed algorithms to distinguish between 10-min inter-seizure versus pre-seizure data clips. Over 10 000 algorithms were submitted. The top algorithms as determined by using the contest data were evaluated on a much larger held-out dataset. The data and top algorithms are available online for further investigation and development. The top performing contest entry scored 0.81 area under the classification curve. The performance reduced by only 6.7% on held-out data. Many other teams also showed high prediction reproducibility. Pseudo-prospective evaluation demonstrated that many algorithms, when used alone or weighted by circadian information, performed better than the benchmarks, including an average increase in sensitivity of 1.9 times the original clinical trial sensitivity for matched time in warning. These results indicate that clinically-relevant seizure prediction is possible in a wider range of patients than previously thought possible. Moreover, different algorithms performed best for different patients, supporting the use of patient-specific algorithms and long-term monitoring. The crowd-sourcing ecosystem for seizure prediction will enable further worldwide community study of the data to yield greater improvements in prediction performance by way of competition, collaboration and synergism.10.1093/brain/awy210_video1awy210media15817489051001.


Subject(s)
Epilepsy/physiopathology , Forecasting/methods , Seizures/physiopathology , Adult , Algorithms , Brain/diagnostic imaging , Brain/physiopathology , Crowdsourcing/methods , Electroencephalography/methods , Female , Humans , Middle Aged , Predictive Value of Tests , Prospective Studies , Reproducibility of Results
7.
ACS Macro Lett ; 7(8): 1010-1015, 2018 Aug 21.
Article in English | MEDLINE | ID: mdl-35650954

ABSTRACT

Biocompatible antibody-nanoparticle conjugates have attracted interest as anticancer agents due to their potential to selectively target therapeutic agents at disease sites. However, new formulation and conjugation approaches are urgently needed to improve their uniformity for clinical applications. Here, a pH-responsive benzaldehyde-functionalized poly[oligo(ethylene glycol) methacrylate-st-para-formyl phenyl methacrylate]-b-poly[2-(diisopropyl)aminoethyl methacrylate] [P(OEGMA-st-pFPMA)-b-PDPA] block copolymer, prepared by reversible addition-fragmentation chain transfer polymerization, produced PEGylated nanoparticles (pH ∼ 7.4) by a single emulsion-solvent evaporation formulation approach. Efficient site-specific attachment of an aminooxy-functionalized anti-EGFR single-domain antibody (sdAb) on these benzaldehyde-decorated nanoparticles is achieved by oxime bond formation. These nanoconjugates can specifically bind EGFR (modified ELISA) and have enhanced uptake over nonfunctionalized controls in EGFR-positive HeLa cells. Encapsulation of rhodamine 6G dye and its dispersion upon cellular uptake, consistent with nanoparticle stability loss at pH < 5.7, prove their ability to facilitate triggered release in endosomal compartments and highlight their potential for use as next-generation antibody-drug nanoconjugates for therapeutic drug delivery.

8.
J Biol Chem ; 292(47): 19238-19249, 2017 11 24.
Article in English | MEDLINE | ID: mdl-28972180

ABSTRACT

Huntington's disease (HD) is caused in large part by a polyglutamine expansion within the huntingtin (Htt) protein. Post-translational modifications (PTMs) control and regulate many protein functions and cellular pathways, and PTMs of mutant Htt are likely important modulators of HD pathogenesis. Alterations of selected numbers of PTMs of Htt fragments have been shown to modulate Htt cellular localization and toxicity. In this study, we systematically introduced site-directed alterations in individual phosphorylation and acetylation sites in full-length Htt constructs. The effects of each of these PTM alteration constructs were tested on cell toxicity using our nuclear condensation assay and on mitochondrial viability by measuring mitochondrial potential and size. Using these functional assays in primary neurons, we identified several PTMs whose alteration can block neuronal toxicity and prevent potential loss and swelling of the mitochondria caused by mutant Htt. These PTMs included previously described sites such as serine 116 and newly found sites such as serine 2652 throughout the protein. We found that these functionally relevant sites are clustered in protease-sensitive domains throughout full-length Htt. These findings advance our understanding of the Htt PTM code and its role in HD pathogenesis. Because PTMs are catalyzed by enzymes, the toxicity-modulating Htt PTMs identified here may be promising therapeutic targets for managing HD.


Subject(s)
Huntingtin Protein/metabolism , Huntington Disease/pathology , Mitochondria/pathology , Mutation , Neurons/pathology , Protein Processing, Post-Translational , Cells, Cultured , Humans , Huntingtin Protein/genetics , Huntington Disease/genetics , Huntington Disease/metabolism , Mitochondria/metabolism , Neurons/metabolism , Phosphorylation
9.
J Proteome Res ; 16(8): 2692-2708, 2017 08 04.
Article in English | MEDLINE | ID: mdl-28653853

ABSTRACT

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington's disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.


Subject(s)
Huntingtin Protein/metabolism , Protein Processing, Post-Translational , Animals , Brain/metabolism , Brain Chemistry , Corpus Striatum/pathology , Humans , Huntingtin Protein/chemistry , Huntington Disease/pathology , Mass Spectrometry/methods , Mice , Nerve Tissue Proteins/metabolism , Peptide Hydrolases/chemistry , Phosphorylation , Protein Domains
10.
Neuron ; 92(6): 1220-1237, 2016 Dec 21.
Article in English | MEDLINE | ID: mdl-27916455

ABSTRACT

Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT.


Subject(s)
Cerebral Cortex/drug effects , Huntington Disease/physiopathology , Neostriatum/drug effects , Phosphodiesterase Inhibitors/pharmacology , Pyrazoles/pharmacology , Quinolines/pharmacology , Animals , Basal Ganglia/diagnostic imaging , Basal Ganglia/drug effects , Basal Ganglia/metabolism , Basal Ganglia/physiopathology , Cerebral Cortex/diagnostic imaging , Cerebral Cortex/metabolism , Cerebral Cortex/physiopathology , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Disease Models, Animal , Huntington Disease/metabolism , Mice , Neostriatum/diagnostic imaging , Neostriatum/metabolism , Neostriatum/physiopathology , Phosphoric Diester Hydrolases , Positron-Emission Tomography , Subthalamic Nucleus/diagnostic imaging , Subthalamic Nucleus/drug effects , Subthalamic Nucleus/metabolism , Subthalamic Nucleus/physiopathology , Tritium
11.
Nat Neurosci ; 19(4): 623-33, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26900923

ABSTRACT

To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo.


Subject(s)
Gene Regulatory Networks/genetics , Genomics/methods , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Proteomics/methods , Animals , Cerebral Cortex/pathology , Cerebral Cortex/physiology , Corpus Striatum/pathology , Corpus Striatum/physiology , Female , Gene Knock-In Techniques/methods , Huntingtin Protein , Male , Mice , Mice, Inbred C57BL
12.
Pain ; 137(1): 182-201, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18160218

ABSTRACT

Neuroinflammatory and neuroimmune mechanisms, as exemplified by infiltrating immune cells and activation of resident endothelial/glial cells, respectively, are known to be involved in the establishment and maintenance of chronic pain. An immune system pathway that may be involved in the activation of both immune and glial cells is complement. The complement pathway is made up of a large number of distinct plasma proteins which react with one another to opsonize pathogens and induce a series of inflammatory responses to help fight infection. Cleaved products and complexes produced by complement activation are responsible for a range of effects including mediation of immune infiltration, activation of phagocytes, opsonization/lysis of pathogens and injured cells, and production of vasoactive amines such as histamine and serotonin. Gene-expression microarray-analysis performed on the rat spinal nerve ligation (SNL) model of neuropathic pain revealed that multiple complement components including the C1 inhibitor, C1q alpha, beta, and gamma, C1r, C1s, C2, C3, C4, C7, and factors B, D, H, and P, were up-regulated while DAF was down-regulated. Regulation of C3 and DAF was confirmed by real-time RT-PCR and in situ hybridization. To test the hypothesis that complement plays a role in neuropathic pain, SNL rats were treated with cobra venom factor (CVF) to deplete plasma of complement component C3. Pain behavior was significantly attenuated in SNL rats treated with CVF as was complement activity at the ipsilateral dorsal root ganglia. Our results suggest the complement pathway might be a novel target for the development of neuropathic pain therapeutics.


Subject(s)
Complement Activation/physiology , Disease Models, Animal , Neuralgia/immunology , Neuralgia/physiopathology , Spinal Nerves/physiology , Animals , Complement Activation/genetics , Ligation , Neuralgia/genetics , Oligonucleotide Array Sequence Analysis/methods , Peripheral Nervous System/immunology , Peripheral Nervous System/pathology , Peripheral Nervous System/physiology , Rats , Spinal Nerves/immunology , Spinal Nerves/pathology
13.
Biochem Pharmacol ; 72(6): 770-82, 2006 Sep 14.
Article in English | MEDLINE | ID: mdl-16844100

ABSTRACT

The N-type voltage-gated calcium channel (Ca(v)2.2) functions in neurons to regulate neurotransmitter release. It comprises a clinically relevant target for chronic pain. We have validated a calcium mobilization approach to assessing Ca(v)2.2 pharmacology in two stable Ca(v)2.2 cell lines: alpha1(B), alpha2delta, beta(3)-HEK-293 and alpha1(B), beta(3)-HEK-293. Ca(v)2.2 channels were opened by addition of KCl and Ca(2+) mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR(96)). Ca(v)2.2 expression and biophysics were confirmed by patch-clamp electrophysiology (EP). Both cell lines responded to KCl with adequate signal-to-background. Signals from both cell lines were inhibited by omega-conotoxin (ctx)-MVIIa and omega-conotoxin (ctx)-GVIa with IC(50) values of 1.8 and 1nM, respectively, for the three-subunit stable, and 0.9 and 0.6nM, respectively, for the two-subunit stable. Other known Ca(v)2.2 blockers were characterized including cadmium, flunarizine, fluspirilene, and mibefradil. IC(50) values correlated with literature EP-derived values. Novel Ca(v)2.2 pharmacology was identified in classes of compounds with other primary pharmacological activities, including Na(+) channel inhibitors and antidepressants. Novel Na(+) channel compounds with high potency at Ca(v)2.2 were identified in the phenoxyphenyl pyridine, phenoxyphenyl pyrazole, and other classes. The highest potency at Ca(v)2.2 tricyclic antidepressant identified was desipramine.


Subject(s)
Calcium Channels, N-Type/metabolism , Calcium/metabolism , Signal Transduction/physiology , Calcium Channel Blockers/pharmacology , Calcium Channels, N-Type/drug effects , Calcium Channels, N-Type/genetics , Cells, Cultured , Electrophysiology , Humans
14.
J Neurophysiol ; 94(5): 3357-67, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16000529

ABSTRACT

Previous studies have shown that layer V pyramidal neurons projecting either to subcortical structures or the contralateral cortex undergo different morphological and electrophysiological patterns of development during the first three postnatal weeks. To isolate the determinants of this differential maturation, we analyzed the gene expression and intrinsic membrane properties of layer V pyramidal neurons projecting either to the superior colliculus (SC cells) or the contralateral cortex (CC cells) by combining whole cell recordings and single-cell RT-PCR in acute slices prepared from postnatal day (P) 5-7 or P21-30 old mice. Among the 24 genes tested, the calcium channel subunits alpha1B and alpha1C, the protease Nexin 1, and the calcium-binding protein calbindin were differentially expressed in adult SC and CC cells and the potassium channel subunit Kv4.3 was expressed preferentially in CC cells at both stages of development. Intrinsic membrane properties, including input resistance, amplitude of the hyperpolarization-activated current, and action potential threshold, differed quantitatively between the two populations as early as from the first postnatal week and persisted throughout adulthood. However, the two cell types had similar regular action potential firing behaviors at all developmental stages. Surprisingly, when we increased the duration of anesthesia with ketamine-xylazine or pentobarbital before decapitation, a proportion of mature SC cells, but not CC cells, fired bursts of action potentials. Together these results indicate that the two populations of layer V pyramidal neurons already start to differ during the first postnatal week and exhibit different firing capabilities after anesthesia.


Subject(s)
Aging/physiology , Anesthetics/administration & dosage , Gene Expression Regulation, Developmental/physiology , Neocortex/physiology , Nerve Tissue Proteins/metabolism , Neurons/physiology , Pyramidal Cells/physiology , Action Potentials/drug effects , Action Potentials/physiology , Aging/drug effects , Animals , Animals, Newborn , Dose-Response Relationship, Drug , Gene Expression Regulation, Developmental/drug effects , Mice , Neocortex/drug effects , Nerve Net/drug effects , Nerve Net/physiology , Neurons/drug effects , Pyramidal Cells/drug effects
15.
J Biomol Screen ; 10(4): 365-73, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15964938

ABSTRACT

A fluorescent imaging plate reader (FLIPR) membrane potential (V(m)) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V(m) response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [(3)H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT(2) inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V(m) assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).


Subject(s)
Amino Acid Transport Systems, Neutral/drug effects , Benzamides/analysis , Membrane Potentials , Spectrometry, Fluorescence/instrumentation , Amino Acid Transport Systems, Neutral/genetics , Animals , Base Sequence , Benzamides/pharmacology , Cell Line , DNA Primers , Glycine Plasma Membrane Transport Proteins , Humans , Rats
16.
J Neurophysiol ; 88(3): 1318-27, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12205153

ABSTRACT

Nicotinic acetylcholine receptors are widely expressed in the neocortex but their functional roles remain largely unknown. Here we investigated the effect of nicotinic receptor activation on interneurons of layer I, which contains a high density of cholinergic fiber terminals. Ninety-seven of 101 neurons recorded in whole cell configuration in rat acute slices were excited by local pressure application of nicotinic agonists, acetylcholine (500 microM), 1,1-dimethyl-4-phenyl-piperazinium (500 microM) or choline (10 mM). Biocytin labeling confirmed that our sample included different morphological types of layer I interneurons. The responses to nicotinic agonists persisted in presence of glutamate and muscarinic receptor antagonists and on further addition of Cd(2+) or tetrodotoxin, indicating that they were mediated by direct activation of postsynaptic nicotinic receptors. The kinetics of the currents and their sensitivity to nicotinic receptor antagonists, methyllycaconitine (1-10 nM) or dihydro-beta-erythroidine (500 nM), suggested that early and late components of the responses were mediated by alpha7 and non-alpha7 types of receptors. Both components had inwardly rectifying I-V curves, which differed when intracellular spermine was omitted. Single-cell RT-PCR experiments identified alpha4, alpha7, and beta2 as the predominantly expressed mRNAs, suggesting that the receptors consisted of alpha7 homomers and alpha4beta2 heteromers. Finally, selective excitation of layer I interneurons through activation of their nicotinic receptors resulted in a tetrodotoxin-sensitive increase of inhibitory synaptic currents recorded in nonpyramidal cells but not in pyramidal cells of layer II/III. These results suggest that acetylcholine released in layer I may induce a disinhibition of the cortical network through activation of nicotinic receptors expressed by layer I interneurons.


Subject(s)
Interneurons/physiology , Neocortex/physiology , Receptors, Nicotinic/physiology , Animals , Electrophysiology , In Vitro Techniques , Interneurons/cytology , Neocortex/cytology , Nicotinic Agonists/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptors, Nicotinic/classification , Receptors, Nicotinic/genetics , Synapses/drug effects , alpha7 Nicotinic Acetylcholine Receptor
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